Gupta R C, Kranias E G
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Biochemistry. 1989 Jul 11;28(14):5909-16. doi: 10.1021/bi00440a030.
A Ca2+-calmodulin-dependent protein kinase was purified to apparent homogeneity from the cytosolic fraction of canine myocardium, with phospholamban as substrate. Purification involved sequential chromatography on DEAE-cellulose, calmodulin-agarose, DEAE-Bio-Gel A, and phosphocellulose. This procedure resulted in a 987-fold purification with a 5.4% yield. The purified enzyme migrated as a single band on native polyacrylamide gels, and it exhibited an apparent molecular weight of 550,000 upon gel filtration. Gel electrophoresis under denaturing conditions revealed a single protein band with Mr 55,000. The purified kinase could be autophosphorylated in a Ca2+-calmodulin-dependent manner, and under optimal conditions, 6 mol of Pi was incorporated per mole of 55,000-dalton subunit. The activity of the enzyme was dependent on Ca2+, calmodulin, and ATP.Mg2+. Other ions which could partially substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations were Sr2+ greater than Mn2+ greater than Zn2+ greater than Fe2+. The substrate specificity of the purified Ca2+-calmodulin-dependent protein kinase for cardiac proteins was determined by using phospholamban, troponin I, sarcoplasmic reticulum membranes, myofibrils, highly enriched sarcolemma, and mitochondria. The protein kinase could only phosphorylate phospholamban and troponin I either in their purified forms or in sarcoplasmic reticulum membranes and myofibrils, respectively. Exogenous proteins which could also be phosphorylated by the purified protein kinase were skeletal muscle glycogen synthase greater than gizzard myosin light chain greater than brain myelin basic protein greater than casein. However, phospholamban appeared to be phosphorylated with a higher rate as well as affinity than glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)
从犬心肌细胞溶质部分以受磷蛋白为底物纯化出一种钙调蛋白依赖性蛋白激酶，达到表观均一性。纯化过程包括依次在二乙氨基乙基纤维素、钙调蛋白琼脂糖、二乙氨基乙基生物凝胶A和磷酸纤维素上进行层析。该方法实现了987倍的纯化，产率为5.4%。纯化后的酶在天然聚丙烯酰胺凝胶上迁移为单一条带，凝胶过滤显示其表观分子量为550,000。变性条件下的凝胶电泳显示一条分子量为55,000的单一蛋白条带。纯化的激酶可在钙-钙调蛋白依赖性方式下进行自身磷酸化，在最佳条件下，每摩尔55,000道尔顿亚基可掺入6摩尔无机磷酸。该酶的活性依赖于钙离子、钙调蛋白和ATP·Mg²⁺。在镁离子存在且钙调蛋白浓度饱和时可部分替代钙离子的其他离子为锶离子＞锰离子＞锌离子＞亚铁离子。通过使用受磷蛋白、肌钙蛋白I、肌浆网膜、肌原纤维、高度富集的肌膜和线粒体，确定了纯化的钙-钙调蛋白依赖性蛋白激酶对心脏蛋白的底物特异性。该蛋白激酶只能分别使纯化形式的受磷蛋白和肌钙蛋白I磷酸化，或在肌浆网膜和肌原纤维中使其磷酸化。也可被纯化蛋白激酶磷酸化的外源蛋白有骨骼肌糖原合酶＞砂囊肌球蛋白轻链＞脑髓鞘碱性蛋白＞酪蛋白。然而，受磷蛋白磷酸化的速率和亲和力似乎均高于糖原合酶。（摘要截短至250字）
